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Cell Biology
Cell Viability, Proliferation & Death
Cell Membrane
Cell Signaling
cell viability, proliferation & death
bacterial cell growth: absorbance
The ssu locus plays a key role in organosulfur metabolism in Pseudomonas putida S-313
J. Bacteriol. 182: 2869–78 (2000).
Antje Kahnert1, Paul Vermeij1, Claudia Wietek1, Peter James2, Thomas Leisinger1, and Michael A. Kertesz1,3.
1 Institute of Microbiology, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich, Switzerland.
2 Protein Chemistry Laboratory, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich, Switzerland.
3 School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
Methods: cell growth assays. Cell growth experiments were performed at 30 °C in microtiter plates containing 150 µL of culture, using a SpectraMax Plus microtiter plate reader with SoftMax Pro software (Molecular Devices), as previously described. The turbidity at 650 nm was measured every 5 minutes, and the plate was shaken for a period of 30 seconds before each measurement.
bacterial cell labeling: fluorescence
Field-scale evaluation of CFDA/SE staining coupled with multiple detection methods for assessing the transport of bacteria in situ
FEMS Microbiology Ecology. 37: 55-66 (2001)
Mark E. Fuller1, Brian J. Mailloux2, Pengfei Zhang3, Sheryl H. Streger1, James A. Hall2, Simon N. Vainberg1, Andrew J. Beavis4, William P. Johnson3, Tullis C. Onstott2, Mary F. DeFlaun1
1Envirogen, Inc. Princeton Research Center, 4100 Quakerbridge Road, Lawrenceville, NJ 08648
2Department of Geosciences, Princeton University, Princeton, NJ 08544
3Department of Geology and Geophysics, University of Utah, Salt Lake City, UT 84112
4Flow Cytometry Core Facility, Department of Molecular Biology, Princeton University, Princeton, NJ 08544.
Background: Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. 5-(and 6-) carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least twenty-eight days as determined by epifluorescent microscopy and flow cytometry. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-105 CFDA/SE-stained cells/mL; the detection limit for flow cytometry was on the order of 1000 cells/mL. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between aqueous cell concentrations determined using the microplate assay and aqueous cell concentrations determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.
Methods: bacterial cell analysis. Enumeration of CFDA/SE-stained cells was performed using a Molecular Devices Gemini (Molecular Devices Corp.) Thirty-five microliters of sample was pipetted into black OptiPlates (Packard Instrument Company), and the fluorescence (Ex 495 nm, Em 538 nm, cutoff 530 nm) of each sample was measured. A standard curve relating the stained cell population (based on direct counts using epifluorescent microscopy of the stained cells prior to transport to the field) and fluorescence was used to convert sample fluorescence to CFDA/SE-stained cells/mL. The limit of detection in the field was 7 x 104 stained cells/mL.
animal cell growth & viability: absorbance
MTT reduction — a tetrazolium-based colorimetric assay for cell survival and proliferation
Molecular Devices MaxLine Application Note #5
Joan M. Chapdelaine
Pharmakon Research International, Inc., Waverly, PA, 18471.
Summary. The MTT assay is a quantitative colorimetric assay for mammalian cell survival and cell proliferation. It depends on the reduction of the tetrazolium salt MTT (3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide) by the mitochondrial dehydrogenase of viable cells to form a blue formazan product. The assay measures cell respiration, and the amount of formazan produced is proportional to the number of living cells present in culture. The assay has been shown to be a simple, rapid alternative to counting cells by dye inclusion/ exclusion, monitoring the release of 51Cr from lysed cells, or the incorporation of [3H]-thymidine into cellular DNA. The MTT assay has been used with a growing number of cell types including primary cultured cells as well as established cell lines. This colorimetric microplate assay is cost effective because of the number of tests which can be performed at one time without the problem of radioisotope and contaminated materials disposal.
Methods: MTT assay. The plates were read at 570 nm with a 630 nm reference to negate the effect of cell debris and precipitated proteins which may be produced by the acidic alcohol addition.
A new quantitative nitroblue tetrazolium reduction assay based on kinetic colorimetry
J. Clin. Lab Anal. 4: 86–89 (1990)
Gabriel Virella, Tab Thompson, and Rebecca Haskill-Strowd.
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425 USA.
Authors report a new quantitative method to follow neutrophil stimulation. The kinetic, colorimetric assay follows the reduction of nitroblue tetrazolium (NBT) in a microplate. The assay is conducted along standard conditions in a microplate, and the color change corresponding to NBT reduction is monitored for 25 minutes at 490 nm. NBT reduction is shown to be clinically significant. NBT reduction values at 1 x 107 cells/mL in normal individuals ranged from 2.59 to 7.41 (4.73 ± 1.89) mOD/min. While patients presenting with symptoms suggestive of chronic granulomatous disease were 0.31 mOD/min. The authors report that this method is considerably simpler than any alternative method for the performance of quantitative NBT assays.
An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT
J. Immunol. Methods 142: 257–265 (1991).
Neal W. Roehm, George H. Rodgers, Stephen M. Hatfield, and Andrew L. Glasebrook.
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285 USA.
Cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines were evaluated using a new chromogenic tetrazolium salt, XTT (sodium 3´-[1-[(phenylamino)-carbonyl]-3, 4-tetrazolium]-bis (4-methoxy-6-nitro)benzene-sulfonic acid hydrate). Reduction of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored, water soluble formazan product obviating the need for crystal solubilization prior to absorbance measurements. The reduction of XTT by the murine cells examined was not particularly efficient. However, addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN) potentiates the reduction of XTT. The combination of XTT/PMS generates higher formazan absorbance values as compared to values with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). XTT is read at 450 nm versus 570 nm for MTT and both use 650 nm as a reference. Authors report the use of XTT in colorimetric proliferation assays offers significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.
animal cell death: absorbance
Identification of the discontinuous epitope in human complement protein C9 recognized by anti-melittin antibodies
J. Immunol. 143: 553–7 (1989)
Roney O. Laine and Alfred F. Esser.
Laboratory of Structural Biology, Department of Comparative and Experimental Pathology, and the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610.
Methods: hemolytic assays. Inhibition of C9-mediated lysis of RBC by the anti-peptides was assayed with EAC1-7 as indicator cells prepared as described. Hemolysis was measured by monitoring turbidity at 650 nm in 96-well microtiter plates (Falcon Microtest III) in a VMax microplate reader (Molecular Devices Corp.) In each sample well 50 µL containing 2.5 x 107 EAC1-7 in GVB-M (5 mM Veronal (barbital), 145 mM NaCl, 0.1% (w/v) gelatin plus 0.15 mM CaCl2, 0.5 mM MgCl2 pH 7.4) and 70 fmol of purified C8 were incubated together at ~4 °C before addition of C9 or C9 preincubated with anti-peptide. Anti-peptide, anti-melittin or nonimmune IgG (30 nmol) was incubated with 70 pmol C9 for 15 minutes at room temperature before mixing with the EAC1-8 cells and adjusting the total well volume to 190 µL with GVB-M, if necessary. The microtiter plates were then incubated at 37 °C, and A650 was measured at predetermined time intervals in the microplate reader. The time required to lyse 50% of the cells (t50%) was used to compare the effects of different antibodies on C9-mediated lysis.
animal cell proliferation: fluorescence
Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin
Proc. Natl. Acad. Sci. USA 98: 6656–6661 (2001).
Tammy L. Moser1, Daniel J. Kenan1, Timothy A. Ashley1, Julie A. Roy1, Michael D. Goodman1, Uma K. Misra1, Dennis J. Cheek, and Salvatore V. Pizzo1.
1 Department of Pathology and Duke University School of Nursing, Duke University Medical Center, Durham, NC 27710.
Methods: cell proliferation assay. Cell density was measured after 24 hours by using the CyQUANT® Cell Proliferation Assay Kit (Molecular Probes) in the Gemini fluorescence microplate reader (Molecular Devices Corp.)
Using the CyQUANT Cell Proliferation Assay Kit in the ƒmax microplate fluorometer
Molecular Devices MaxLine Application Note #23.
This application note details two methods for using the CyQUANT Cell Proliferation Assay Kit with the ƒmax microplate fluorometer. In the first method, cellular proliferation is quantitated using a cell-based standard curve. In the second method, cellular proliferation is quantitated using RNAse treated cell samples and a DNA standard curve. The data in this application note were obtained using an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Excitation and emission filters of these wavelengths are included as standard equipment with the ƒmax microplate fluorometer. Molecular Probes describes a dynamic range from 50 to at least 50,000 cells for the standard assay in a microplate format. The kit contains sufficient reagent for 1000 assays using a 200 µL-per-well volume. The data presented here are preliminary and do not represent a fully optimized assay.
LIVE/DEAD® Viability/Cytotoxicity Kit assay for animal cells using the Gemini XS fluorescence microplate reader
Molecular Devices MaxLine Application Note #43.
Anne T. Ferguson, Ph.D.
Methods: LIVE/DEAD Viability/Cytotoxicity assay. The LIVE/DEAD Viability/Cytoxicity Kit (Molecular Probes, cat# L-3224) was used with Chinese Hamster Ovary (CHO) cells in 96-well black microtiter plates with clear bottoms (Costar, cat# 3603). CHO cells were grown in Ham’s F12 medium containing 10% FBS. Cells were washed three times with PBS and then treated for 20 minutes with 3 mL 0.526 mM EDTA in PBS. The detached cells were harvested and diluted in PBS. An aliquot was counted using a hemocytometer. The concentration of cells was adjusted to 2.5 x 105 cells/mL in PBS, which is equivalent to a starting concentration of 2.5 x 104 cells/100 µL. For preparation of dead cells, half of the cells were treated with 1:100 dilution of 10% Triton X-100 for 10 minutes. Both live and dead cells were serially diluted 1:3 in PBS to obtain a range of concentrations from 2.5 x 104 to 103 cells/100 µL. Cells were plated such that each column of the microplate had 8 replicates of a live or dead cell dilution. The negative control/background was 100 µL of PBS. A solution of 11.4 µM calcein AM (cal AM)/5.7 µM ethidium homodimer-1 (EthD-1) in PBS was made and 100 µL aliquots were added to each well of the microtiter plate, including the control wells. The final concentration of dyes in each well was 5.7 µM cal AM/2.85 µM EthD-1. Cells were incubated with the dyes for 30 minutes at 37 °C in a tissue culture incubator and then analyzed using the Gemini XS. The optimal instrument settings for cal AM-stained live cells were Ex 485 nm/Em 525 nm using a 515 nm emission cutoff filter and for EthD-1 stained dead cells were Ex 525 nm/Em 620 nm using a 590 nm emission cutoff filter.
Measurement of green fluorescent protein in the Gemini XS spectrofluorometer
Simon Lydford1 and Thomas Giller2.
Molecular Devices MaxLine Application Note #44.
1 Molecular Devices Limited.
2 Axovan Limited.
Methods: GFP assay. Human embryonic kidney 293 cells (HEK-293) were transiently transfected with a wild-type GFP expression plasmid (pS65T, Clontech, discontinued product; cytomegalovirus immediate early promoter drives expression of GFP) using Lipofectamine as per manufacturer's instructions (Gibco/Invitrogen, Cat. No. 18324-012). Cells were grown to confluence in 162 cm2 flasks under standard cell-culture conditions. Fluorescence microscopy revealed an expression level in excess of 75%. The GFP-expressing cells were removed with trypsin, and the cells were washed with medium (MEM Alpha, Gibco-RBL Cat. No. 22571-020). The appropriate number of cells was seeded in alternate columns of a 96-well microplate (Costar, black walled, clear bottomed Cat. No. 3603) and diluted with mock-transfected cells to maintain a constant cell number of 200,000 cells per well. This resulted in a final number of 6,250 (column 12) to 200,000 (column 2) GFP-expressing cells per well in a volume of 100 µL. These cells were seeded in alternate columns 2, 4, 6, 8, 10, and 12. In contrast, columns 1, 3, 5, 7, 9 and 11 were seeded with 200,000 mock-transfected cells per well. Then the plate was returned to the incubator for 48 hours. Initially, excitation and emission scans were run to select the optimum excitation wavelength and emission wavelength/cutoff filter combination for the maximum signal/background ratio. The parameters selected for all subsequent experiments were: GFP; lEX – 472 nm, lEM – 512 nm with a 495 nm emission cutoff filter.
cell membrane
lipid docking assay: fluorescence
Close is not enough: SNARE-dependent membrane fusion requires an active mechanism that transduces force to membrane anchors
J. Cell Biol. 150: 105–117 (2000).
James A. McNew, Thomas Weber, Francesco Parlati, Robert J. Johnston, Thomas J. Melia, Thomas H. Söllner, and James E. Rothman.
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Methods: liposome docking assay. Adherence of the docked liposomes was performed for 1 hour at room temperature on a platform. Undocked v-SNARE–containing liposomes were removed by five 300 µL washes with PBS. The remaining docked, v-SNARE–derived rhodamine fluorescence was determined by solubilizing the remaining lipid in 100 µL of 2% SDS and measuring rhodamine fluorescence (excitation 535 nm, emission 590 nm, emission cutoff at 570 nm) in a fluorescent plate reader (Gemini, Molecular Devices Corp.)
cell adherence: absorbance
2´-Hydroxychalcone inhibits nuclear factor-B and blocks tumor necrosis factor — and lipopolysaccharide-induced adhesion of neutrophils to human umbilical vein endothelial cells
Mol. Pharmacol. 58: 526–34 (2000).
Babita Madan1, Sanjay Batra1, and Balaram Ghosh.
Molecular Immunology and Immunogenetics Laboratory, Centre for Biochemical Technology, University of Delhi Campus (North), Delhi, India.
Methods: cell adherence assay. Adhesion of neutrophils to endothelial monolayers was assayed as described previously (Dobrina et al., 1991). Nonadherent neutrophils were removed by washing the wells with PBS thrice. Adherent neutrophils were assayed colorimetrically by adding a substrate solution (100 µL/well) consisting of o-phenylenediamine dihydrochloride (40 mg/100 mL in citrate phosphate buffer, pH 4.5) containing 0.1% cetitrimethyl ammonium bromide as peroxidase solubilizing agent. The interference by the few contaminating eosinophils was abolished by adding a selective eosinophil peroxidase inhibitor, 3-amino-1,2,4 triazole (1 mM) to the substrate solution. After 2 minutes of incubation, 2N H2SO4 (50 µL/well) was added to stop the reaction. The absorbance was determined at 490 nm using an automated microplate reader (SpectraMax 190; Molecular Devices Corp).
cell signaling
g-protein coupled receptors: luminescence
Ca++-induced G protein-coupled receptor (GPCR) activation monitored via aequorin luminescence in the LMax™ microplate luminometer
Molecular Devices MaxLine Application Note #42 (2000).
Jinfang Liao, M.D., Ph.D. and Evelyn McGown, Ph.D.
This application note demonstrates how to use LMax to detect receptor-activated Ca++ signals in cells stably expressing apo-aequorin. In these cell-based assays, we were able to construct agonist concentration-response curves for the activation of a Gq-protein-coupled purinergic receptor expressed endogenously in CHO cells. Thus, the LMax microplate luminometer can be used to study GPCR pharmacology. Furthermore, with a constant agonist concentration in the injector reservoir, the LMax microplate luminometer could potentially be used to run high-thoughput luminescent assays for GPCR antagonist screening.
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